PROJECT SUMMARY Lower respiratory tract infection (LRTI) frequently contributes to respiratory failure that requires mechanical ventilation, and ventilator-associated pneumonia is a feared complication in patients dependent upon mechanical ventilation. Despite the clinical importance of LRTI in critical care, the dynamics of the respiratory tract microbiome during mechanical ventilation that may correspond with risk for infection and the respiratory tract microbiome features that define LRTI remain poorly understood. The proposed study will utilize deep sequencing of the 16S rRNA gene from upper and lower respiratory tract samples obtained from critically ill, mechanically ventilated subjects in order to improve understanding of healthcare-associated microbiome change, LRTI microbial ecology, and LRTI pathogenesis. The study will (1) define the longitudinal change that occurs in the upper and lower respiratory tract bacterial microbiome during mechanical ventilation; (2) determine the features of respiratory tract microbiome community structure that correlate with the clinical diagnosis of LRTI and with the absence of LRTI; and (3) evaluate the ability of 16S rRNA gene sequencing to identify bacterial pathogens responsible for LRTI. Execution of these specific aims will elucidate the relationships between healthcare exposures and respiratory tract microbiome change, and between respiratory tract microbiome change and LRTI pathogenesis. The proposed study will thus form a foundation for an independent career in patient-oriented research dedicated to improving understanding of healthcare- associated microbiome change and infection. Execution of the aims will also provide the primary investigator with essential experience directing a large, observational microbiome study and with essential training in the advanced statistical and bioinformatics methods necessary to analyze the interaction between patient-level factors (underlying disease, antibiotic exposure, etc), microbial ecology, and infection. The research proposal is paired with a career development plan that makes use of the extensive resources of the University of Pennsylvania, and which capitalizes on a mentorship committee with broad, complementary expertise in infectious diseases epidemiology, healthcare-associated infection, deep sequencing methods, microbiology, and critical care. Dr. Ebbing Lautenbach, Dr. Frederic D. Bushman, and Dr. Ronald G. Collman will support the primary investigator in the outlined path towards a successful career as an independent clinical investigator.